Fluorescence Microscopy Techniques Research Articles

SPITFIR(e): a supermaneuverable algorithm for fast denoising and deconvolution of 3D fluorescence microscopy images and videos

Modern fluorescent microscopy imaging is still limited by the optical aberrations and the photon budget available in the specimen. A direct consequence is the necessity to develop flexible and “off-road” algorithms in order to recover structural details and improve spatial resolution, which is critical when restraining the illumination to low levels in order to limit photo-damages. Here, we report SPITFIR(e) a flexible method designed to accurately and quickly restore 2D–3D fluorescence microscopy images and videos (4D images). We designed a generic sparse-promoting regularizer to subtract undesirable out-of-focus background and we developed a primal-dual algorithm for fast optimization. SPITFIR(e) is a ”swiss-knife” method for practitioners as it adapts to any microscopy techniques, to various sources of signal degradation (noise, blur), to variable image contents, as well as to low signal-to-noise ratios. Our method outperforms existing state-of-the-art algorithms, and is more flexible than supervised deep-learning methods requiring ground truth datasets. The performance, the flexibility, and the ability to push the spatiotemporal resolution limit of sub-diffracted fluorescence microscopy techniques are demonstrated on experimental datasets acquired with various microscopy techniques from 3D spinning-disk confocal up to lattice light sheet microscopy.

A method for reproducible high-resolution imaging of 3D cancer cell spheroids.

Multicellular tumour cell spheroids embedded within three-dimensional (3D) hydrogels or extracellular matrices (ECM) are widely used as models to study cancer growth and invasion. Standard methods to embed spheroids in 3D matrices result in random placement in space which limits the use of inverted fluorescence microscopy techniques, and thus the resolution that can be achieved to image molecular detail within the intact spheroid. Here, we leverage UV photolithography to microfabricate PDMS (polydimethylsiloxane) stamps that allow for generation of high-content, reproducible well-like structures in multiple different imaging chambers. Addition of multicellular tumour spheroids into stamped collagen structures allows for precise positioning of spheroids in 3D space for reproducible high-/super-resolution imaging. Embedded spheroids can be imaged live or fixed and are amenable to immunostaining, allowing for greater flexibility of experimental approaches. We describe the use of these spheroid imaging chambers to analyse cell invasion, cell-ECM interaction, ECM alignment, force-dependent intracellular protein dynamics and extension of fine actin-based protrusions with a variety of commonly used inverted microscope platforms. This method enables reproducible, high-/super-resolution live imaging of multiple tumour spheroids, that can be potentially extended to visualise organoids and other more complex 3D in vitro systems.

Protein Nanofibrils from Fava Bean and Its Major Storage Proteins: Formation and Ability to Generate and Stabilise Foams.

Protein nanofibrils (PNFs) have potential for use in food applications as texture inducers. This study investigated the formation of PNFs from protein extracted from whole fava bean and from its two major storage proteins, globulin fractions 11S and 7S. PNFs were formed by heating (85 °C) the proteins under acidic conditions (pH 2) for 24 h. Thioflavin T fluorescence and atomic force microscopy techniques were used to investigate PNF formation. The foaming properties (capacity, stability, and half-life) were explored for non-fibrillated and fibrillated protein from fava bean, 11S, and 7S to investigate the texturing ability of PNFs at concentrations of 1 and 10 mg/mL and pH 7. The results showed that all three heat-incubated proteins (fava bean, 11S, and 7S) formed straight semi-flexible PNFs. Some differences in the capacity to form PNFs were observed between the two globulin fractions, with the smaller 7S protein being superior to 11S. The fibrillated protein from fava bean, 11S, and 7S generated more voluminous and more stable foams at 10 mg/mL than the corresponding non-fibrillated protein. However, this ability for fibrillated proteins to improve the foam properties seemed to be concentration-dependent, as at 1 mg/mL, the foams were less stable than those made from the non-fibrillated protein.

Functional basis for calmodulation of the TRPV5 calcium channel.

Within the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a highly Ca2+ -selective channel important for active reabsorption of Ca2+ in the kidney. Its channel activity is controlled by a negative feedback mechanism involving calmodulin (CaM) binding. Combining advanced microscopy techniques and biochemical assays, this study characterized the dynamic lobe-specific CaM regulation. We demonstrate for the first time that functional (full-length) TRPV5 interacts with CaM in the absence of Ca2+ , and this interaction is intensified at increasing Ca2+ concentrations sensed by the CaM C-lobe that achieves channel pore blocking. Channel inactivation occurs without requiring CaM N-lobe calcification. Moreover, we show a Ca2+ -dependent binding stoichiometry at the single channel level. In conclusion, our study proposes a new model for CaM-dependent regulation - calmodulation - of this uniquely Ca2+ -selective TRP channel TRPV5 that involves apoCaM interaction and lobe-specific actions, which may be of significant physiological relevance given its role as gatekeeper of Ca2+ transport in the kidney. KEY POINTS: The renal Ca2+ channel TRPV5 is an important player in maintenance of the body's Ca2+ homeostasis. Activity of TRPV5 is controlled by a negative feedback loop that involves calmodulin (CaM), a protein with two Ca2+ -binding lobes. We investigated the dynamics of the interaction between TRPV5 and CaM with advanced fluorescence microscopy techniques. Our data support a new model for CaM-dependent regulation of TRPV5 channel activity with CaM lobe-specific actions and demonstrates Ca2+ -dependent binding stoichiometries. This study improves our understanding of the mechanism underlying fast channel inactivation, which is physiologically relevant given the gatekeeper function of TRPV5 in Ca2+ reabsorption in the kidney.

Imaging neuro-urodynamics of mouse major pelvic ganglion using a micro-endoscopic approach.

Post-ganglionic neurons of the autonomic nervous system lie outside of the central nervous system and innervate specific target effectors such as organs or glands. The major pelvic ganglion (MPG) is one such ganglion that plays a significant role in controlling bladder function in rodents. However, due to technical and physical constraints in recording electrophysiological signals from these neurons in vivo, the functional neural activity in MPG is mostly unknown. Transgenic animal models expressing genetically encoded calcium indicators now provide opportunities to monitor the activity of populations of neurons in vivo to overcome these challenges related to traditional electrophysiological methods. However, like many peripheral neurons, the MPG is not conducive to conventional fluorescent microscopy techniques, as it is located in the pelvic cavity thus limiting robust optical access by benchtop microscopes. Here, we present an endoscopic approach based on a custom miniscope system (UCLA V3) that allows for effective in vivo monitoring of neural activity in the MPG for the first time. We show that our imaging approach can monitor activity of hundreds of MPG neurons simultaneously during the filling and emptying of the bladder in urethane-anesthetized transgenic mouse line expressing GCaMP6s in cholinergic MPG neurons. By using custom analysis scripts, we isolated the activity of hundreds of individual neurons and show that populations of neurons have distinct phasic activation patterns during sequential bladder filling and voiding events. Our imaging approach can be adapted to record activity from autonomic neurons across different organs and systems in both healthy and disease models.

Evaluation of Antifungal Activity and Potential Application as Fluorescent Probes of Indolenine and Benzo[e]Indole-Based Squarylium Dyes.

The antifungal performance and the possible use as fluorescent probes of a series of squarylium dyes derived from indolenine and benzo[e]indole previously synthesized was evaluated. Some photophysical properties were performed in ethanol and phosphate buffer, and the type of aggregates form in phosphate buffer was analyzed. Using the 1,3-diphenylisobenzofuran assay, a qualitative assessment of the capacity of dyes to produce singlet oxygen after irradiation was performed. Regarding the antifungal activity, this was studied through a broth microdilution assay using Saccharomyces cerevisiae PYCC 4072 as a biological model. The effect of irradiation of the dyes, with an appropriate light emitting diode system, on the antifungal activity was also evaluated, and it was verified that some of the dyes improve their activity after irradiation. Using fluorescence microscopy techniques, the colocalization of dyes in S. cerevisae cells was investigated and it was possible to verify that some of the squarylium dyes with a barbituric moiety in the four-membered central ring stained and accumulated preferentially in the mitochondrial web and perinuclear membrane of the cells. The possible use as a fluorescent probe for the detection of HSA was also evaluated for one of the dyes of the series, demonstrating a linear variation in the fluorescence intensity accompanied by the increase in the protein concentration.

Advances in high spatiotemporal resolution fluorescence microscopic imaging technique based on point scanning

Laser point-scanning fluorescence microscopy serves as an indispensable tool in the life science research, owing to its merits of excellent resolution, high sensitivity, remarkable specificity, three-dimensional optical-sectioning capability, and dynamic imaging. However, conventional laser point-scanning fluorescence microscopy confronts a series of challenges in the rapidly evolving field of life sciences, because of the limitations imposed by optical diffraction and point scanning detection. Over the past two decades, substantial advancements have been made in super-resolution fluorescence microscopic imaging techniques. Researchers have developed various high spatial and temporal resolution point-scanning microtechniques, which hold great significance for biological optical imaging and other relevant applications. Regrettably, there are still few review articles covering the recent progress of this field. It is essential to provide a comprehensive review of laser point-scanning fluorescence microscopic techniques for their future developments and trends. In this article, the basic principles and recent advances in different point-scanning fluorescence microscopy imaging techniques are introduced from the perspectives of temporal resolution and spatial resolution, and the progress and applications of high spatio-temporal resolution microscopic imaging techniques based on point-scanning mode are summarized. Finally, the development trends and challenges of high spatio-temporal resolution point scanning fluorescence microscopic imaging technique are discussed.

Anti-proliferative activity of nitroquinolone fused acylhydrazones as non-small cell human lung cancer agents.

A new series of 8-nitroquinolone-based aromatic heterocyclic acyl hydrazones have been synthesised and characterised through various spectroscopic techniques. They were theoretically examined for molecular docking with various proteins related to the apoptosis of the non-small cell lung cancer cell line A549. The results indicate that the possible modes of interaction of all the synthesised compounds are compatible for use as anti-proliferative drugs. Also, the drug-likeness of the compounds was examined through theoretical ADMET analysis, which indicated good gastrointestinal absorption as well as low toxicity. Selected compounds were evaluated for their in vitro anti-cancer activity using A549, MCF-7 and HeLa cell lines through an MTT assay to determine cytotoxicity. Compounds 3c, 3a and 11c exhibited significant cytotoxicity towards A549 cells in the order of 3c (15.3 ± 0.7) > 3a (15.8 ± 0.1) > 11c (17.1 ± 0.2), whereas all the compounds show insignificant toxicity on normal human embryonic kidney cells up to a concentration of 200 μM. The best compounds among the series (3c and 11c) were chosen for further detection of apoptosis through fluorescence microscopic techniques using AO/EtBr and DAPI. The reduced DNA synthesis during the cell cycle was also investigated through flow cytometric techniques. The results indicate that the compounds possess significant anticancer properties due to the activation of the mitochondrial mediated intrinsic pathway.

Advanced fluorescence microscopy in respiratory virus cell biology.

Respiratory viruses are a major public health burden across all age groups around the globe, and are associated with high morbidity and mortality rates. They can be transmitted by multiple routes, including physical contact or droplets and aerosols, resulting in efficient spreading within the human population. Investigations of the cell biology of virus replication are thus of utmost importance to gain a better understanding of virus-induced pathogenicity and the development of antiviral countermeasures. Light and fluorescence microscopy techniques have revolutionized investigations of the cell biology of virus infection by allowing the study of the localization and dynamics of viral or cellular components directly in infected cells. Advanced microscopy including high- and super-resolution microscopy techniques available today can visualize biological processes at the single-virus and even single-molecule level, thus opening a unique view on virus infection. We will highlight how fluorescence microscopy has supported investigations on virus cell biology by focusing on three major respiratory viruses: respiratory syncytial virus (RSV), Influenza A virus (IAV) and SARS-CoV-2. We will review our current knowledge of virus replication and highlight how fluorescence microscopy has helped to improve our state of understanding. We will start by introducing major imaging and labeling modalities and conclude the chapter with a perspective discussion on remaining challenges and potential opportunities.

Applications of fluorescence spectroscopy in protein conformational changes and intermolecular contacts

Emission fluorescence is one of the most versatile and powerful biophysical techniques used in several scientific subjects. It is extensively applied in the studies of proteins, their conformations, and intermolecular contacts, such as in protein-ligand and protein-protein interactions, allowing qualitative, quantitative, and structural data elucidation. This review, aimed to outline some of the most widely used fluorescence techniques in this area, illustrate their applications and display a few examples. At first, the data on the intrinsic fluorescence of proteins is disclosed, mainly on the tryptophan side chain. Predominantly, research to study protein conformational changes, protein interactions, and changes in intensities and shifts of the fluorescence emission maximums were discussed. Fluorescence anisotropy or fluorescence polarization is a measurement of the changing orientation of a molecule in space, concerning the time between the absorption and emission events. Absorption and emission indicate the spatial alignment of the molecule's dipoles relative to the electric vector of the electromagnetic wave of excitation and emitted light, respectively. In other words, if the fluorophore population is excited with vertically polarized light, the emitted light will retain some polarization based on how fast it rotates in solution. Therefore, fluorescence anisotropy can be successfully used in protein-protein interaction investigations. Then, green fluorescent proteins (GFPs), photo-transformable fluorescent proteins (FPs) such as photoswitchable and photoconvertible FPs, and those with Large Stokes Shift (LSS) are disclosed in more detail. FPs are potent tools for the study of biological systems. Their versatility and wide range of colours and properties allow many applications. Finally, the application of fluorescence in life sciences is exposed, especially the application of FPs in fluorescence microscopy techniques with super-resolution that enables precise in vivo photolabeling to monitor the movement and interactions of target proteins.

Research progress of miniature head-mounted single photon fluorescence microscopic imaging technique

The predecessor of China Optics was China Optics and Applied Optics Digest, which was founded in 1985. At that time, it was the only retrieval journal in the field of optics in China. At the end of 2008, China Optics and Applied Optics Digest was renamed

Fluorescence Light Reflection of Different Dental Materials: A Literature Review

Abstract Fluorescence, the emission of light by a material after absorbing photons, is a property that has been extensively studied and utilized in various fields. In dentistry, fluorescence plays a crucial role in assessing the esthetic outcomes of dental restorations and prostheses. Different dental materials, such as resin composites and ceramics, exhibit distinct fluorescence characteristics. Understanding how these materials interact with fluorescent light is essential for achieving optimal esthetics in dental treatments. Therefore, fluorescence light reflection of different dental materials is a topic of great significance in the field of dentistry. Researchers have employed various methods to investigate the fluorescent light reflection of dental materials. These methods include spectrophotometry, fluorescent microscopy, and digital imaging techniques. By analyzing the fluorescence emission spectra and intensity, valuable insights into the optical properties of different dental materials have been obtained. Studies have shown that factors such as material composition, surface roughness, and color stability can influence the fluorescence light reflection of dental materials. For example, resin composites with certain additives or modifications may exhibit enhanced fluorescence, resulting in more natural-looking restorations. Furthermore, advancements in digital imaging technology have allowed for the development of fluorescencebased devices, such as fluorescence imaging cameras, to aid in clinical assessments. These devices enable dentists to evaluate the esthetic integration of dental materials within the oral environment, leading to improved treatment outcomes. Overall, the investigation of fluorescence light reflection of different dental materials offers valuable insights into their optical properties and their potential impact on esthetics. The understanding gained from this research can aid in the selection and characterization of dental materials, ultimately enhancing the esthetic outcomes of dental treatments

Bullseye Analysis: A Fluorescence Microscopy Technique to Detect Local Changes in Intracellular Reactive Oxygen Species (ROS) Production.

Reactive oxygen species (ROS) are naturally produced compounds that play important roles in cell signaling, gene regulation, and biological defense, including involvement in the oxidative burst that is central to the anti-microbial actions of macrophages. However, these highly reactive, short-lived radical species also stimulate cells to undergo programmed cell death at high concentrations, as well as causing detrimental effects such as oxidation of macromolecules at more moderate levels. Imaging ROS is highly challenging, with many researchers working on the challenge over the past 10-15 years without producing a definitive method. We report a new fluorescence microscopy-based technique, Bullseye Analysis. This methodology is based on concepts provided by the FRAP (Fluorescence Recovery after Photobleaching) technique and refined to evidence the spatiotemporal production of ROS, and the subsequent consequences, on a subcellular scale. To exemplify the technique, we have used the ROS-reporter dye, CellROX, and the ROS-inducing photosensitizer, LightOx58, a potent source of ROS compared with UV irradiation alone. Further validation of the technique was carried out using differing co-stains, notably Mitotracker and JC-1.

Quantitative Element-Sensitive Analysis of Individual Nanoobjects.

A reliable and quantitative material analysis is crucial for assessing new technological processes, especially to facilitate a quantitative understanding of advanced material properties at the nanoscale. To this end, X-ray fluorescence microscopy techniques can offer an element-sensitive and non-destructive tool for the investigation of a wide range of nanotechnological materials. Since X-ray radiation provides information depths of up to the microscale, even stratified or buried arrangements are easily accessible without invasive sample preparation. However, in terms of the quantification capabilities, these approaches are usually restricted to a qualitative or semi-quantitative analysis at the nanoscale. Relying on comparable reference nanomaterials is often not straightforward or impossible because the development of innovative nanomaterials has proven to be more fast-paced than any development process for appropriate reference materials. The present work corroborates that a traceable quantification of individual nanoobjects can be realized by means of an X-ray fluorescence microscope when utilizing rather conventional but well-calibrated instrumentation instead of reference materials. As a proof of concept, the total number of atoms forming a germanium nanoobject is quantified using soft X-ray radiation. Furthermore, complementary dimensional parameters of such objects arereconstructed.

Comparative study of pre-Hispanic and colonial adobes in Mexico. Preliminary inferences on the effects of the granulometric distribution and used recycled materials in the state conservation of earth architecture

The characterization of the pre-Hispanic and colonial adobes used in the construction of buildings in Mexico was carried out to know the differences between their processes and manufactures. The eight samples obtained correspond to adobe blocks 0.45 m long x 0.35 m wide and 0.12 m thick, which were part of the interiors of houses, the structure of fences, and hacienda galleys. These samples were investigated using X-ray Diffraction, X-ray Fluorescence, and Scanning Electron Microscopy techniques to determine the morphometry of the material and its chemical and mineralogical composition. Likewise, some analyzes were performed to determine their mechanical properties (simple compressive strength), physical properties (granulometry, solid density, cohesion, plasticity index, porosity, and moisture content), and biological (organic matter content). The results obtained in terms of the granulometric distribution indicate that pre-Hispanic adobe has a higher content of sand than silt-clay and gravel, while the colonial adobes present a low or null content of gravel with a higher content of silt-clays. Regarding the organic matter content, the colonial adobe presents the highest percentages compared to those of the pre-Hispanic adobes. Taking into account the above the pre-Hispanic adobes have better resistance and durability than colonial adobes due to their granulometric distribution, low organic matter content, good cohesion, high plasticity index, and the use of materials found in their natural environment, while the colonial used recycled materials, coming from the economic activity to which the hacienda was dedicated, for example, agriculture, mining, livestock, this has given rise to different manufacturing techniques and construction styles that reflect the state of conservation of buildings throughout the pre-Hispanic and colonial times. The granulometric distribution that pre-Hispanic adobe provides favorable physical and mechanical qualities for any construction technique, so it is recommended to use this formula to manufacture resistant and durable adobe.

Bacto_tracker: a method for single-cell tracking of M. xanthus in dense and multispecies colonies

Cell motility and predation are important for the dynamics of many multi-cellular ecosystems, such as the gut or the soil. Approaches to image cell dynamics in such complex systems are scant, and high-throughput analysis methods to segment and track single-cell behaviors are currently lacking. Here, we addressed these limitations by implementing a fast fluorescence microscopy technique enabling the high-resolution acquisition of cell movement over large areas and long time periods. Next, we applied deep learning to semantically segment two different bacteria species within complex micro-environments . We implemented a method to build single cell traces by combining the cell masks from different time points to follow the dynamics of single cells with high spatial and temporal resolutions and over long periods of time. We applied and validated these methods by characterizing the dynamics of Escherichia coli predation by Myxococcus xanthus.

Evidence and Mass Quantification of Atmospheric Microplastics in a Coastal New Zealand City.

This study investigated the atmospheric deposition of microplastics (MPs) in Auckland, New Zealand, from two sampling sites over a 9-week period. The sizes, morphologies, number counts, and mass concentrations of specific polymers were determined for airborne MPs using a combination of a Nile Red-assisted automated fluorescence microscopy technique in series with pyrolysis-gas chromatography-mass spectrometry (Pyr-GC/MS). This enabled a larger number of MPs to be analyzed from each sample compared to traditional spectroscopic techniques. Microplastic number concentrations increased exponentially with decreasing size. The results show the importance of using consistent methodologies and size cutoffs when comparing microplastic data between studies. Eight polymers were quantified in the atmospheric deposition samples, with polyethylene (PE), polycarbonate (PC), and poly(ethylene terephthalate) (PET) being the most commonly observed. The largest MP deposition rates at an urban rooftop correlated with winds originating from the marine environment with speeds between 15 and 20 m s-1, indicating that airborne MPs in coastal regions may originate from wave-breaking mechanisms. This study represents the first report of using Pyr-GC/MS to determine the chemical compositions and mass concentrations of atmospheric microplastics, along with corresponding data on their sizes, morphologies, and number counts.

Single Plane Illumination Microscopy for Microfluidic Device Imaging

Three-dimensional imaging of live processes at a cellular level is a challenging task. It requires high-speed acquisition capabilities, low phototoxicity, and low mechanical disturbances. Three-dimensional imaging in microfluidic devices poses additional challenges as a deep penetration of the light source is required, along with a stationary setting, so the flows are not perturbed. Different types of fluorescence microscopy techniques have been used to address these limitations; particularly, confocal microscopy and light sheet fluorescence microscopy (LSFM). This manuscript proposes a novel architecture of a type of LSFM, single-plane illumination microscopy (SPIM). This custom-made microscope includes two mirror galvanometers to scan the sample vertically and reduce shadowing artifacts while avoiding unnecessary movement. In addition, two electro-tunable lenses fine-tune the focus position and reduce the scattering caused by the microfluidic devices. The microscope has been fully set up and characterized, achieving a resolution of 1.50 μm in the x-y plane and 7.93 μm in the z-direction. The proposed architecture has risen to the challenges posed when imaging microfluidic devices and live processes, as it can successfully acquire 3D volumetric images together with time-lapse recordings, and it is thus a suitable microscopic technique for live tracking miniaturized tissue and disease models.

Suitability of lectin binding studies for the characterization of redox-active microbial environmental biofilms

Aquatic environmental microbial biofilms grow in a broad range of redox environments from oxic to methanogenic, and they often also establish internal redox gradients. In technical applications, biofilms are also subjected to controlled redox conditions. Studies on biofilms often make use of fluorescence microscopic imaging techniques together with lectin binding analysis to gain insights into structure, composition, and functions of the biofilms. Here we studied the direct influence of redox potentials on fluorescence lectin binding analyses (FLBA) for two commonly used lectin-fluorophore conjugates. An effect of the electrical potential on signal intensity was observed and found to be statistically significant. The signal intensity changes however, remained within the range of a few percent total. A significant drop in intensity was only observed for extremely oxidizing potentials, typically not found under environmental conditions. Our results showed that the fluorophore itself and not the lectin binding to the respective glycoconjugate causes fluorescence changes. The two tested lectin-fluorophores are shown to be suitable for studying the distribution and composition of EPS in environmental biofilms or technical applications and under varying redox conditions.

The use and detection of quantum dots as nanotracers in environmental fate studies of engineered nanoparticles

Investigations of the behavior and effects of engineered nanoparticles (ENPs) on human health and the environment need detailed knowledge of their fate and transport in environmental compartments. Such studies are highly challenging due to low environmental concentrations, varying size distribution of the particles and the interference with the natural background. A strategy to overcome these limits is to use mimics of ENPs with unique detectable properties that match the properties of the ENPs as nanotracers. A special class of ENPs that can be tracked are quantum dots (QDs). QDs are composed of different metals, metalloids, or more recently also carbon (e.g., graphene), that result in unique optical properties. This allows the tracking of such particles by fluorescence microscopic and photometric techniques. Many types of QDs consist of heavy elements, allowing to track and visualize these particles also by electron microscopy and to quantitate the particles indirectly based on these elements. QDs can also be surface modified in various ways which enable them to be used as a label or as traceable mimics for ENPs. This review reflects a broad range of methods to synthesize and modify QDs based on metals, metalloids, and graphene for studying the environmental fate of nanoparticles and discusses and compares analytical methods that can be used for tracking and quantifying QDs. In addition, we review applications of QDs as ENP mimics in environmental studies of surface waters, soils, microorganisms, and plants with respect to the applied analytical techniques.